Chapter 3: Methods
3.1 Method and Theory
Most produced biodiesel worldwide is currently achieved through alkaline- catalyzed methanolysis. However, in this process, the oils feedstock needs to be refined to remove free fatty acids, which not only increase the processing cost but also lead to loss of part of the feedstock [148, 149]. Lipase-catalyzed production on the other hand, has drawn increasing attention, since it can be effectively used unrefined oil, in addition to its other advantages, including the operation at mild conditions, low energy requirements and easy product separation [150].
For economical application of enzymatic processed in biodiesel production, lipase enzyme has to be used in immobilized form to allow easy retention and reuse.
In addition, immobilization has been reported to result in enhanced enzyme thermal and shear stability [148, 153]. The main challenges facing industrialization of enzymatic process are the high mass transfer resistance, tendency to adsorb the by- product glycerol onto the support matrix and poor operational stability [150]. These problems can be solved by good selection of supports of favorable surface characteristics and pore sizes [154, 155].Porous supports are generally preferred due to their high surface area, which allows a higher loading and better protection of the enzyme [154]. Therefore the pore size plays an important role on the catalytic activity and stability [156, 157, 158].
In that regard, increasing interest has recently been on Metal-Organic Frameworks (MOFs) as a new kind of porous supports for enzyme immobilization [159, 160]. In addition, it was shown that by immobilizing peroxidase and trypsin on
MOF composites higher stability and catalytic performance was attained [159].
Physical adsorption, which is a fast and easy method of enzyme immobilization ensures limited enzyme denaturation and does not affect the enzyme activity, native structure and active sites [160]. However, owing to the weak interaction between the enzyme and the support, with this method enzymes are prone to leaching, which results in low stability [161]. Hierarchical mesoporous (ZIF-8) was also used to immobilize Burkholderia cepacia lipase (BCL) by surface adsorption, and in biodiesel production [162]. A biodiesel yield of 93.4% was achieved after 12 h with three-step addition of alcohol at 40°C showed the immobilization efficiency was shown to depend on the adsorption time, immobilization temperature, pH, and morphology of ZIF‐8. As an alternative, chemisorption in which the nucleophiles of the enzymes (free amino acids) are covalently bonded to the organic linkers (mainly carboxylate groups) of MOFs to form peptide bonds has been used [163]. This result in more stable biocatalysts, with less enzyme leaching and more rigid backbone [150, 164, 165].
However, the negative effect of the chemical bonds on the structure of the enzyme renders physically immobilized lipase generally more active,, despite the higher operational stability of chemically immobilized lipase [161]. Having said that, it should be noted that with both adsorption approaches, enzyme attachment into the internal pores of MOFs may not be efficient, as part of the enzymatic activity may be lost due to conformational changes during diffusion into small cavities [150, 158].
To reduce the leaching problem encountered by physical adsorption, while avoiding the chemical adsorption which could negatively affect the activity of the enzyme, mesoporous MOFs, such as MIL‐100(Fe) and HKUST‐1, have been used as supports for lipase immobilization through co‐precipitation [154, 166]. In this case, the enzyme is caged inside the pores of the MOFs during the crystallization process.
As the immobilization is presumably non-covalent, so minor activity loss against that of the free enzyme could be reached Lipase encapsulated inside MOFs was successfully used in transesterification reaction and showed higher thermal stability than free lipase [158]. However, owing to the long‐range ordering and nonuniformity of MOFs, low enzyme loadings were achieved. Lipase was firstly in situ encapsulated inside ZIF-8 and ZIF-10 for biosensing of explosive organic-peroxides [158], Candida antarctica lipase B was also encapsulated inside UiO-66 and ZIF-8 and used for the transesterification of vinyl acetate and vinyl laurate [168]. By using encapsulated lipase inside ZIF-67, 78% biodiesel yield was achieved after 60h at 45oC [176]. A higher biodiesel yield of 92.3% was achieved within 24 h using Rhizomucor miehei lipase encapsulated in X-shaped ZIF-8 at 40oC. In addition, the enzyme retained 84.7%
of its initial activity after 10 repeated cycles [169]. In addition, enzymes immobilized by this approach exhibit mass transfer limitations and their diffusion is restricted as the substrate may not be able to access the entire active sites [170]. Kinetics and thermodynamics studies of the adoption process, which is scarce in literature, could provide invaluable information on the surface chemical affinity, , enzyme accessibility and leaching [149]. Therefore, in this work, the mechanism, kinetics and thermodynamics of lipase adsorption on the surface of different MOFs, namely ZIF- 67, ZIF-8 and HKUST-1, have been thoroughly investigated and tested for biodiesel production. The three supports have different structures, pore sizes, chemical properties, and surface areas.
Adsorption is a process that results in the removal of a solute from a solution and concentrating it at the surface of the adsorbent, until the amount of the solute remaining in the solution is in equilibrium with that at the surface. In this work, the assumption based that after 24 hr of batch reaction will reach an equilibrium[174].
In this work, the mechanism, kinetics and thermodynamics of lipase adsorption on the surface of different MOFs, namely ZIF-67, ZIF-8 and HKUST-1, have been thoroughly investigated and tested for biodiesel production. The three supports have different structures, pore sizes, chemical properties, and surface areas.
Many theoretical and empirical models have been developed to represent the various types of adsorption isotherms. At present, there is no single model that satisfactory describes all mechanisms and shapes. Langmuir and Freundlich models, described by Equations (1) and (2) have been widely used to describe adsorption isotherms [174, 175, 172]:
𝑞𝑒 = 𝑞𝑚𝑏𝐶𝑒
1 + 𝑏𝐶𝑒 (1)
𝑞𝑒 = 𝑎𝐹𝐶𝑒1/𝑏 (2)
Where, qe is the equilibrium amount of solute adsorbed (mg/g of solid), Ce is the equilibrium concentration of solute in solution (mg/L), qm (mg/g) and b (mg/L−1) are constants, representing the maximum adsorption capacity for the solid phase loading and the energy constant related to the heat of adsorption, respectively, and aF (mg(1 −1/b) l1/b/g) and b are constants.
The Langmuir isotherm assumes uniform and constant binding of the sorbate on the surface of the adsorbent. The Freundlich model does not have thermodynamic basis and does not offer physical interpretation of the adsorption data.
The value for the Langmuir isotherm constant (b) determined at different temperatures can be used to calculate the enthalpy change of adsorption
thermodynamic parameters, such as Gibbs free energy (ΔG), change in enthalpy (ΔH), and entropy (ΔS), as given by Equation (3) [172]:
𝑙𝑛𝑏 = −∆𝐺
𝑅𝑇= −∆𝐻 𝑅𝑇 +∆𝑆
𝑅 (3)
Where T is the temperature (K), R is the gas constant (8.314 J/mol K), and b is the Langmuir constant. Adsorption kinetics on the other hand, describes the reaction pathways and the time needed to reach the equilibrium. In order to examine the controlling mechanism, kinetics models that have been commonly used are the Lagergren pseudo-first order, Elovich’s model and Pseudo-Second order and intraparticle diffusion models. The linearized form of those models is given in Equations (4-7), and in Table 3 respectively [172]:
𝑙𝑛(𝑞𝑒− 𝑞) = 𝑙𝑛(𝑞𝑒) − 𝑘𝑡
2.303 (4)
𝑞 =1
𝑏𝑙𝑛(𝑎𝑏) +1
𝑏𝑙𝑛(𝑡) (5)
𝑡 𝑞= 1
𝑘2𝑞𝑡2+ 1
𝑞𝑒𝑡 (6)
𝑞𝑡= 𝐶 + 𝑘𝑖𝑑𝑡0.5 (7)
Where, k is the kinetics constant of pseudo-first order adsorption (min−1), qe and q (mg/g) are the amounts adsorbed at equilibrium and at time t (min), respectively, a is the initial adsorption rate (mg/g/min), and 1/b (mg/g) is a parameter related to the number of sites available for adsorption, k2 (g/mg/min) is the pseudo-second order rate constant of adsorption, kid is the rate constant of the intraparticle transport
(mg/g/min0.5) and C (mg/g) is a constant related to the thickness of the boundary layer.
The higher the value of C, the greater boundary layer effect.
Table 3: Rearranging of linear kinetic models’ parameter’s
Type Linear Form Plot Parameter
Pseudo Second order
model
𝑡 𝑞𝑡 = 1
𝑘𝑞𝑒2+ 1
𝑞𝑒𝑡 t/qt vs. t
qe= 1/slope k=slope2/Intercept
Pseudo First
order model 𝑙𝑛(𝑞𝑒− 𝑞) = 𝑙𝑛(𝑞𝑒) − 𝑘𝑡
2.303 ln(qe-q) vs t
qe=e(Intercept)
k= -Slope x 2.303
Elovich’s
model 𝑞 =1
𝑏𝑙𝑛(𝑎𝑏) +1
𝑏𝑙𝑛(𝑡) Q vs. ln(t)
b=1/slope Intercept = slope x ln(1/slope x a)
The rate limiting step in the Pseudo-Second order is the surface adsorption that involves chemisorption, where the adsorbate removal from a solution is due to physicochemical interactions between the two phases [174]. Wherein, Pseudo first order model assumes intraparticle transport (pore diffusion) rate-limiting step [175].
In the intraparticle diffusion model assumed the process to be diffusion- controlled, and the rate of adsorption depends on the speed at which adsorbate diffuses towards adsorbent [174].The intraparticle diffusion coefficients can be determined using Equations (8)[152]:
𝐷𝑝 = 0.03 × 𝑟𝑝2⁄𝑡𝑒0.5 (8)
Where, DP is the intraparticle diffusion (cm2/s), rP is the average radius obtained from BET (nm) and te is the contact time required to reach the equilibrium (min)